High Performance Liquid Chromatographic Determination of Acidic Catecholamine Metabolites in Human Urine Using Postcolumn Fluorescence Derivatization with dl-1, 2-Diphenylethylenediamine

Abstract

dl-1, 2-Diphenylethylenediamine was found to react sensitively in an alkaline solution with 3, 4-dihydroxymandelic acid as well as other catechol compounds and 4-hydroxy-3-methoxyphenyl compounds to yield fluorescent products. By using this reaction, a postcolumn fluorescence derivatization high performance liquid chromatography of the acidic metabolites of catecholamines (3, 4-dihydroxymandelic acid, 3, 4-dihydroxyphenylacetic acid, vanillylmandelic acid and homovanillic acid) in human urine was established. These compounds were separated on an anion-exchanger column (TSK-gel DEAE-2SW) and subjected to derivatization involving coulometric oxidation followed by a fluorescence reaction with the named reagent. The detection limits (S/N=3) for the acidic metabolites in urine were 15-30fmol in a 100-μl injection volume.